(T1030-03-15) Coarse-Grained Molecular Dynamic Simulations of Protein-Membrane System

Purpose: The purpose of this study was to investigate how the structural and electrostatic properties of coiled-coil peptides influence their cell-penetrating capabilities, with the goal of informing more effective therapeutic delivery systems. Using coarse-grained molecular dynamics (CG-MD) simulations and in vitro experiments, the study systematically compared two variants: the positively charged K coil ((VSALKEK)₄) and the negatively charged E coil ((VSALEKE)₄). The study aimed to elucidate the mechanistic role of proteins in membrane interaction through electrostatic attraction to phospholipid bilayer (cell membrane), while also evaluating functional outcomes by conjugating these peptides to fluorescent proteins (RFP and GFP) in cell culture models. By correlating computational predictions of peptide-lipid bilayer adsorption with experimental observations of intracellular protein delivery efficiency, this work provides a framework for optimizing CPP design through strategic balancing of amphipathicity and charge distribution.

Methods: 1) Structure Prediction: The amino acid sequences of the coiled-coil peptides (K-coil: (VSALKEK)₄ ; Ecoil: (VSALEKE)₄) were submitted to AlphaFold3 to generate 3D structural models. 2) Membrane-Peptide System Setup: The Martini force field (v3) was used for CG-MD, employing 4 to 1, 3 to 1 and 2 to 1 mapping scheme with chemically specific bead types to model peptide-lipid interactions efficiently. The predicted structures were imported into CHARMM-GUI’s Membrane Builder. Initially the protein is converted into a CG-Model using martinize2.py script. The cell membrane was modeled using a mixture of 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (anionic) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (neutral). Two peptides either K-coil or E-coil were positioned above the upper leaflet of a pre equilibrated lipid bilayer composed of 512 lipids (256 per leaflet, POPG:POPC ratio 1:1). The system was solvated with Martini water molecules and neutralized with 0.15 M NaCl while maintaining electrostatic neutrality. 3) Simulation Protocol: Energy Minimization: Performed using the steepest descent algorithm (25,000 steps) to remove steric clashes and optimize the system geometry. Equilibration with Position Restraints: Conducted in the NPT ensemble (310.15 K, 1 bar) for 20 ns with stepwise relaxation of restraints. Production Run: Unrestrained MD simulation performed for 1 μs (20 fs timestep) under NPT conditions (310.15 K, 1 bar). Visualization, trajectory and Radial Distribution Function (RDF) analysis were performed by VMD and energy analysis were performed by GROMACS. 4) In-Vitro Studies: K coil was fused to mCherry, and E coil was fused to EGFP, each with a C-terminal His-tag. Both constructs were cloned into the pET21 vector. Recombinant vector were transformed in E.coli cells. Protein expression was induced for 4 hours at 32°C. The proteins were purified using nicked affinity chromatography and subsequently dialyzed in 20 mM Tris HCl pH 7.4 buffer and 150 mM NaCl buffer. Proteins were individually incubated with A549 cells to check for their internalization using fluorescence microscope.

Results: 1) Structure prediction: The structure predicted by AF3 showed high confidence level proving the folding to be accurate. 2) Molecular Dynamic Simulation: Visual analysis revealed successful integration of the K coil into the upper leaflet of the lipid bilayer. Full translocation across the bilayer is a macroscopic process, requiring extended simulation timescales and energy considerations beyond the current study. Both proteins initially resided 36–40 Å above the bilayer center. By ~200 ns, the K coil stabilized within the bilayer, while the E coil failed to achieve stability throughout the 1000-ns simulation Despite containing both glutamic acid (E) and lysine (K) residues, the E coil’s net negative charge (due to E residue dominance) created electrostatic repulsion with anionic lipid headgroups, overriding hydrophobic stabilization. 3) Interaction Energy Analysis: K Coil  a) Lennard-Jones (L-J): Strong interactions driven by hydrophobic residues (Val, Leu)   b) Coulombic: Favorable interactions between lysine (K) residues and anionic PO₄⁻ groups (POPG, POPC). E Coil   a) Repulsive Coulombic forces (E residues vs. PO₄⁻) dominated, preventing effective van der Waals (hydrophobic) interactions at short distances. 4) Radial Distribution Function (RDF): The K coil showed preferential binding to POPG lipids: Strongest interaction with PO₄⁻ groups of POPG. Moderate interaction with glycerol backbones. Minimal interaction with positively POPC choline groups (due to charge repulsion). 5) Cellular Uptake: The K coil facilitated intracellular delivery of mCherry (26 kDa) into A549 cells, as confirmed by fluorescence microscopy. The E coil failed to internalize EGFP, consistent with MD simulation outcomes.

Conclusion: The K-coil successfully interacted with the lipid membrane, consistent with supporting in-vitro data. The E-coil, lacking positive charge, did not exhibit similar interactions, highlighting its inability to associate with the membrane. Results demonstrate that positive charge and hydrophobic residues are critical for effective peptide-membrane interactions. MD simulations proved to be a valuable tool for understanding protein-lipid interactions and can guide the design of next-generation CPP.

References: 1) Li, J.; Tuma, J.; Han, H.; Kim, H.; Wilson, R. C.; Lee, H. Y.; Murthy, N. The Coiled-Coil Forming Peptide (KVSALKE)5 Is a Cell Penetrating Peptide That Enhances the Intracellular Delivery of Proteins. Adv. Healthc. Mater. 2022, 11 (9), e2102118. https://doi.org/10.1002/adhm.202102118.
2) Abramson, J.; Adler, J.; Dunger, J.; Evans, R.; Green, T.; Pritzel, A.; Ronneberger, O.; Willmore, L.; Ballard, A. J.; Bambrick, J.; Bodenstein, S. W.; Evans, D. A.; Hung, C.-C.; O’Neill, M.; Reiman, D.; Tunyasuvunakool, K.; Wu, Z.; Žemgulytė, A.; Arvaniti, E.; Beattie, C.; Bertolli, O.; Bridgland, A.; Cherepanov, A.; Congreve, M.; Cowen-Rivers, A. I.; Cowie, A.; Figurnov, M.; Fuchs, F. B.; Gladman, H.; Jain, R.; Khan, Y. A.; Low, C. M. R.; Perlin, K.; Potapenko, A.; Savy, P.; Singh, S.; Stecula, A.; Thillaisundaram, A.; Tong, C.; Yakneen, S.; Zhong, E. D.; Zielinski, M.; Žídek, A.; Bapst, V.; Kohli, P.; Jaderberg, M.; Hassabis, D.; Jumper, J. M. Accurate Structure Prediction of Biomolecular Interactions with AlphaFold 3. Nature 2024, 630 (8016), 493–500. https://doi.org/10.1038/s41586-024-07487-w.
3) Jo, S.; Kim, T.; Iyer, V. G.; Im, W. CHARMM-GUI: A Web-Based Graphical User Interface for CHARMM. J. Comput. Chem. 2008, 29 (11), 1859–1865. https://doi.org/10.1002/jcc.20945.
4) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1–2, 19–25. https://doi.org/10.1016/j.softx.2015.06.001.
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Fig. 1. Protein Structure predicted by AF3
plDDT - predicted local distance difference test
Fig. 2. CG Mapping of K-Coil
Fig. 3. All Atom Model of POPG and POPC Lipids
Coarse Grain Model of POPC and POPG Lipids



Fig. 4. All molecules are in VDW representation. Protein in yellow and pink color, water in green. (A) Initial System for K-Coil and Lipid Bilayer System (B) Initial System without Water Molecules for better visualization
(C) Top View of the Initial System without water

Fig. 4. Protein in yellow and pink color with licorice representation, POPG head shown in cyan and POPC head shown in blue with VDW representation. All other molecules are hidden for better visualization (A) Final System of K-Coil and Lipid Bilayer System after 1µs Simulation (B) Top view of K-Coil and Lipid Bilayer System (C) Final System of E-Coil and Lipid Bilayer System after 1µs Simulation (D) Top view of E-Coil and Lipid Bilayer System.



Fig. 5. (A) Center of Mass Separation Distance in the Z-Direction Between the Protein and the Lipid Membrane over time
(B) Lennard-Jones Interactions over time
(C) Electrostatic Interactions over time
(D) RDFs of lipid groups with K-coil